Geoff Mealing

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-d-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol‐rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen–glucose deprivation (OGD), N‐methyl‐d‐aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only β‐, methylated β‐ and sulfated β‐CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD‐95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X‐100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated β‐CD to protect against NMDA toxicity, and reversed NR2B, PSD‐95 and nNOS localization to Triton X‐100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor‐mediated increases in intracellular Ca2+ concentration ([Ca2+]i), but did suppress OGD‐induced increases in [Ca2+]i. β‐CD, but not Mβ‐CD, also caused a slight block of NMDA‐induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent‐resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol‐rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca2+ levels.

Angiotensin II‐induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol‐phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris‐phosphate (IP3), intracellular calcium, [Ca2+]i, and protein kinase C (PKC), in angiotensin II (AII)‐induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP3 and a rapid increase in [Ca2+]i in cells loaded with the Ca2+‐responsive fluorescent dye fura‐2. The prompt AII‐induced [Ca2+]i spike was not affected by incubating HCEC in Ca2+‐free medium containing 2 mM EGTA or by pretreating the cultures with the Ca2+ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca2+ from intracellular stores. The AII‐triggered increases in IP3, [Ca2+]i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar1, Val5, Ala8‐AII (SVA‐AII), and by the phospholipase C (PLC) inhibitors, neomycin and U‐73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII‐induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood‐brain barrier permeability in acute hypertension, is likely dependent on PLC‐mediated changes in [Ca2+]i and independent of PKC.

Attenuation of neurotoxicity in cortical cultures and hippocampal slices from E2F1 knockout mice

The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and β‐amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological oxygen‐glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(+/+) and (−/−) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration ([Ca2+]i) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(−/−) cultures were significantly less susceptible to neuronal death than E2F1(+/+) cultures 24 and 48 h following 120–180 min of OGD. Furthermore, the absence of E2F1 significantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our finding that E2F1 mRNA levels are significantly increased following OGD, support a role for E2F1 in the modulation of OGD‐ and anoxia‐induced neuronal death. These findings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.

Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-d-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

N-methyl-D-aspartate- or glutamate-mediated toxicity in cultured rat cortical neurons is antagonized by FPL 15896AR

Abstract: The neuroprotective action of (S)‐α‐phenyl‐2‐pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 µM) or glutamate (50 µM) for 15 min resulted in the death of 85–95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 µM) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 µM) prevented the loss of membrane‐associated protein kinase C activity that developed by 4 h after transient exposure to 50 µM NMDA or 50 µM glutamate. FPL 15896AR also reduced by ∼35% the magnitude of NMDA‐triggered increases in intracellular free Ca2+ concentration in the cortical cultures. These data indicate that NMDA‐mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.

A fluorescence confocal assay to assess neuronal viability in brain slices.

Hippocampal slice models are used to study the mechanisms of ischemia-induced neurotoxicity and to assess the neuroprotective potential of novel therapeutic agents. A number of morphological and functional endpoints are available to assess neuronal viability. The slice model also allows the study of selectively vulnerable neuronal populations within the same preparation. The fluorescence procedure described here provides a method of assessing the viability of neurons in rat hippocampal slices exposed to hypoxic-hypoglycemic conditions. Control and/or treated slices that had been subjected to a 10 min oxygen-glucose deprivation insult are double stained with calcein-AM (4 microM), which stains live cells green, and ethidium homodimer (6 microM), which stains the nucleus of dead cells red. The stained slices are then imaged using confocal microscopy. Vulnerable neurons in the CA1 region of slices deprived of oxygen and glucose became increasingly permeant to ethidium homodimer over the 4 h reperfusion period. Exposure to low Ca2+ concentration (0.3 mM) or the N-, P- and Q-type Ca2+ channel antagonist MVIIC (100 nM), which have been shown to be neuroprotective in this model of ischemia using field evoked post-synaptic potential (EPSP) measures as an endpoint, were also shown to be protective using the fluorescence assay.

An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD‐active MnIIIporphyrins meso‐substituted with N, N′‐dimethylimidazolium or N‐alkylpyridinium groups did not protect neurons against oxygen‐glucose deprivation (OGD), although lower SOD‐active and ‐inactive para isomers protected against N‐methyl‐d‐aspartate (NMDA) exposure. MnIIImeso‐tetrakis(4‐benzoic acid)porphyrin (MnIIITBAP), as well as SOD‐inactive metalloTBAPs and other phenyl ring‐ or β‐substituted metalloporphyrins that contained redox‐insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD‐ or NMDA‐induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor‐mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD‐inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600.

Cell placement and guidance on substrates for neurochip interfaces.

Interface devices such as integrated planar patch‐clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly‐d‐lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo‐preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch‐clamp interfaces on the chip. Biotechnol. Bioeng. 2010; 105: 368–373.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip’s polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)—polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.